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Method for purifying deoxyribonucleic acid (DNA) in DNA bisulfate conversion process

A bisulfite and conversion process technology, applied in the field of DNA purification, can solve the problems of decreased recovery rate, time-consuming, unsatisfactory processing effect, etc., and achieve the effects of shortening processing time, increasing sensitivity, and improving quality

Active Publication Date: 2014-09-10
TIANGEN BIOTECH BEIJING
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Problems solved by technology

However, under such conditions, a large number of active oxygen groups will be generated in the reaction system, which will fragment and degrade DNA, thus reducing the sensitivity of the converted PCR and subsequent analysis techniques, and also The recovery rate of DNA is reduced, sometimes even less than 50%
[0004] Based on the above-mentioned characteristics of bisulfite conversion, the traditional conversion method usually optimizes the conversion conditions relatively mildly to ensure the integrity of DNA, but under such mild treatment conditions, researchers will sacrifice a lot of processing time, so It will cause the whole processing process to be very cumbersome and time-consuming, generally taking about 18 hours
But even so, for precious trace samples (such as 1ng), the degradation of DNA is still inevitable, so the processing effect of traditional methods is not satisfactory

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  • Method for purifying deoxyribonucleic acid (DNA) in DNA bisulfate conversion process

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Embodiment

[0049] Four human genome samples of 10 microliters (the amount of DNA is 1000 nanograms) were respectively processed by the method of the present invention, and the processing effect of the samples was detected by subsequent methylation-specific PCR. The specific operation steps are as follows:

[0050] (1) Add 10 microliters of DNA solution to be treated, 10 microliters of DNA protection agent and 90 microliters of DNA bisulfite conversion solution to a 200 microliter centrifuge tube, and make up the reaction volume to 120 microliters with deionized water Microliter;

[0051] The formula of the DNA bisulfite conversion solution described therein is: tetraethylammonium chloride: 1.5 grams, bisulfite: 0.4 grams, weigh the two components and dissolve them in deionized water, and use sodium hydroxide to solution to adjust the pH value of the mixed solution to 5.5, and finally dilute the solution to 1000 ml with deionized water;

[0052] The DNA protecting agent described therei...

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Abstract

The invention relates to a method for purifying deoxyribonucleic acid (DNA) in a DNA bisulfate conversion process and belongs to the technical field of nucleic acid conversion and purification. According to the method disclosed by the invention, a DNA protecting agent capable of preventing DNA from fragmenting and degrading is added to a DNA bisulfate conversion system, so that the DNA is not fragmented or degraded in the treatment process of bisulfate. Thus, the quality of the processed DNA is effectively improved; the yield of nucleic acid is about 70%; and the sensitivity of polymerase chain reaction (PCR) and other analysis technologies is improved. By adopting the method disclosed by the invention, the entire treatment process of the DNA bisulfate is simpler and faster; and meanwhile, the quality of the processed DNA is improved.

Description

technical field [0001] The invention relates to a method for purifying DNA in the DNA bisulfite conversion process, which is beneficial to speeding up the reaction speed in the bisulfite conversion process and protecting the integrity of DNA, and belongs to the technical field of nucleic acid conversion and purification. Background technique [0002] Although there are many methods for detecting DNA methylation, among many methods, researchers will first use a technique, which is the bisulfite conversion of DNA. Bisulfite conversion of DNA converts unmethylated cytosines in DNA to uracil, while methylated cytosines remain unchanged. Therefore, it is possible to determine which bases in the DNA sequence to be tested are methylated by comparing the DNA sequences before and after treatment by subsequent analysis methods such as sequencing, methylation-specific PCR or chips. [0003] Although this conversion is simple in theory, the actual bisulfite conversion is not so easy to...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
Inventor 张双宇俞萍李晓晨孙克非
Owner TIANGEN BIOTECH BEIJING
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